PROJECT ABSTRACT The proposed Bay Area Team Against Resistance U54 Project (BATAR-UP) is an interdisciplinary effort of investigators to apply their knowledge and expertise to dissect the molecular and cellular basis of incomplete response and resistance to current treatments and to identify new treatment strategies to better neutralize or eliminate residual disease and prevent resistance. This translational approach will be part of the NCI's Drug Resistance and Sensitivity Centers Network to develop innovative strategies to understand and combat mechanisms of tumor resistance and exploit tumor sensitivity to anti-cancer therapies. To accomplish this, BATAR-UP will support two projects and one core driven by a multidisciplinary team of investigators at UCSF and Stanford University. Project 1 will define and interrogate the molecular and cellular basis of residual disease in lung cancers treated with targeted inhibitors in clinical use. We will prioritize for initial study both EGFR-mutant and ALK gene rearrangement positive lung cancers, given their importance as key molecular disease subtypes and our prior published work and expertise. We will harness genetic and transcriptomic analysis of clinical samples (liquid and tumor biopsies) to provide a molecular view of the evolution of response, residual disease, and acquired resistance. We will generate organoid and PDX models and apply cutting-edge functional screens (genetic, pharmacologic, and targeted proteomic assays) to identify key vulnerabilities that could be therapeutically exploited, including with CTEP agents. This systematic approach will allow us to reveal the basis of the incomplete response and residual disease that drives EGFR and ALK inhibitor resistance and pinpoint therapeutic strategies to intercept the evolution of residual disease and eventual acquired resistance. Project 2 will define and interrogate the molecular and cellular basis of resistance and residual disease in lung cancers treated with current immunotherapies, including PD-1 and PD- L1 antibodies. Leveraging shared platforms in synergy with Project 1, we will perform systematic analyses of liquid and tumor biopsy specimens (and ex vivo models) obtained from patients longitudinally before and during treatment and upon acquired resistance. We will focus our studies on EGFR and ALK wild type patients, including squamous cell lung cancer and adenocarcinoma patients where immunotherapy has shown efficacy but is typically non-curative. We will leverage (1) a novel lung cancer organoid model wherein tumor biopsies are cultured as both tumor epithelium and their endogenous tumor infiltrating lymphocytes (TILs) en bloc as a cohesive unit, and (2) deep droplet-based single-cell RNA-seq analysis. Our systematic approach will help define the basis of the incomplete response and residual disease that contributes to immunotherapy resistance and identify potential new therapeutic strategies to help convert these incomplete responses into curative outcomes. Our Administrative Core will provide leadership, coordination and oversight for BATAR-UP with the overarching goal of synergizing the research conducted in the 2 Projects and the entire DRSC network.